Primary cell-based high-throughput screening for identification of compounds that antagonize MrgX1 receptor signaling
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Kaiping Xu M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 3868
Data Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Xinzhong Dong, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA033176-01
Grant Proposal PI: Xinzhong Dong, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Kaiping Xu M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D.
Name: Primary cell-based high-throughput screening for identification of compounds that antagonize MrgX1 receptor signaling
Mrgs are a family of orphan G protein-coupled receptors (GPCRs) which are specifically expressed in subsets of small-diameter sensory neurons in dorsal root ganglia (DRG) as these genes have not been detected in the central nervous system or in the rest of the body. Primary sensory neurons in DRG play an essential role in generating pain and itch by detecting painful or itchy stimuli through their peripheral axons in the skin and sending signals to the spinal cord via their central axons.
Several complementary lines of evidence suggest that Mrgs function as endogenous inhibitors of persistent pain . Particularly, spinal cord application of BAM8-22 in mice strongly inhibited the wind-up response of wide dynamic range (WDR) neurons in the spinal cord, and significantly attenuated inflammatory hyperalgesia and neuropathic pain in an Mrg-dependent manner. BAM8-22, a 15 amino acid endogenous peptide, is a specific agonist for mouse MrgC11 and human MrgX1 [2, 3]. Agonists[4,5] for Mrgs may represent a new class of anti-hyperalgesics for chronic pain, which could suppress central sensitization via peripheral modulation, thereby inhibiting chronic pain without side effects in the central nervous system. Interestingly, the dual effect of BAM8-22 (i.e. inducing itch in the skin and blocking chronic pain in the spinal cord) is analogous to that of morphine (i.e. blocking pain and inducing itch).
Itch, also known as pruritus, is defined as an "unpleasant skin sensation that elicits the desire or reflex
to scratch" . Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch by detecting itch stimuli through their peripheral axons in the skin and sending signals to the spinal cord via their central axons . The best characterized itch mediator is histamine, which is mainly secreted by skin mast cells and excites nearby sensory fibers by activating histamine receptors . Histamine-induced itch in human skin is almost completely blocked by histamine H1 receptor antagonists. However, the blockers are ineffective in more than two-thirds of itch conditions, such as those arising from atopic dermatitis, psoriasis, renal and liver diseases, drug treatment, poisonous plants and mechanical stimuli . These observations along with other electrophysiological and molecular studies indicate the existence of histamine-independent forms of itch [9-11]. Despite the clinical importance of process, the cellular and molecular mechanisms of histamine-independent itch remain largely undefined. The lack of identified cell surface receptors that directly respond to histamine independent itch stimuli is currently the major hurdle to developing novel anti-pruritus therapeutics.
The major goal of this project is to carry out a large-scale screen of small molecular compounds for antagonists, agonists, and allosteric modulators of human MrgX1 with therapeutic implications of anti-itch and anti-chronic pain.
Principle of the assay
The principle of the assay is based on that MrgX1 is potently and specifically activated by BAM8-22 (specific peptide agonist) which evokes a large rise in intracellular Ca2+. A three-addition scheme has been established for the multiplex screening of different kinds of modulators in one screen by three additions: 1) 1st addition for drug (library) itself for agonists, as well as an intrinsic counter screen to overcome common non-specificity of the calcium flux assays. 2) 2nd addition: addition of EC10 to EC30 of BAM8-22 for screen of allosteric agonist. 3) 3rd addition: addition of EC90 to ECmax of BAM8-22 for screen of antagonists/allosteric antagonists.
The purpose of this assay is to identify test compounds that act as an antagonist for MrgX1. This assay employs a HEK293 cell line that stably expresses MrgX1 protein. The cells, which loaded with fluorescent dye-Flou4 NW, are treated with test compounds, followed by measurement of calcium flux.. Those HEK293 cells stably expressing MrgX1 were plated into 384-well plates. On the following day, cells were incubated with Fluo4-NW solution at 37 C for 30min and at RT for 30. min after removing media. 10 uM Compounds were added to the assay buffer with dye and recorded the change of fluorescence by Hamamatsu FDSS 6000 kinetic imaging plate reader. Compound effect was evaluated by the calculated fluorescence ratio. If the compound causes more than 3 times the standard deviation of the B-scores of the library compounds, the compound is then considered to be active as an agonist of the MrgX1 protein.
MrgX1, BAM 8-22, GPCR, itch, pain, chronic pain, HTS assay, counter, HEK293, 384, primary, Antagonist, allosteric, agonist, FDSS, Kinetic, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Guan, Y., et al., Mas-related G-protein-coupled receptors inhibit pathological pain in mice. Proc Natl Acad Sci U S A, 2010. 107(36): p. 15933-8. PMID:20724664
2. Han, S.K., et al., Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated byRF-amide-related peptides through the Galpha q/11 pathway. Proc Natl Acad Sci U S A, 2002. 99(23):p. 14740-5. PMID:12397184
3. Lembo, P.M., et al., Proenkephalin A gene products activate a new family of sensory neuron-specific GPCRs. Nat Neurosci, 2002. 5(3): p. 201-9. PMID:11850634
4. Malik, L., et al., Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis. Bioorg Med Chem Lett, 2009. 19(6): p. 1729-32. PMID:19230660
5. Wroblowski, B., et al., The discovery of a selective, small molecule agonist for the MAS-related gene X1 receptor. J Med Chem, 2009. 52(3): p. 818-25. PMID:19146417
6. Ikoma, A., et al., The neurobiology of itch. Nat Rev Neurosci, 2006. 7(7): p. 535-47. PMID:16791143
7. Paus, R., et al., Frontiers in pruritus research: scratching the brain for more effective itch therapy. J Clin Invest, 2006. 116(5): p. 1174-86. PMID:16670758
8. Alving, K., et al., Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs. Cell Tissue Res, 1991. 264(3): p. 529-38. PMID:1714354
9. Davidson, S., et al., The itch-producing agents histamine and cowhage activate separate populations of primate spinothalamic tract neurons. J Neurosci, 2007. 27(37): p. 10007-14. PMID:17855615
10. Johanek, L.M., et al., A role for polymodal C-fiber afferents in nonhistaminergic itch. J Neurosci, 2008. 28(30): p. 7659-69. PMID:18650342
11. Johanek, L.M., et al., Psychophysical and physiological evidence for parallel afferent pathways mediating the sensation of itch. J Neurosci, 2007. 27(28): p. 7490-7. PMID:17626210
12. Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 1999. 4(2),67-73. PMID: 10838414.
13. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175. PMID: 16465162.
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0 for the 3rd addition
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors .
10) Calculate B scores  for test compounds using ratios calculated in Step 8.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (<=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, AND not as a MrgX1 agonist in 1st addition, the compound is designated in the Outcome as active as an antagonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.
List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)